T cells mediate most forms of cellular immunity, including cell lympholysis, delayed type hypersensitivity (DTH), transplantation rejection, and allograft rejection. An introduction to T cells and cell mediated immunity is found in Paul (1993) Fundamental Immunology, Third Edition Raven Press, New York, N.Y. and the references cited therein.
Typical T cells do not respond to free antigenic peptides. Instead, T cells interact with a specialized set of cell surface proteins (the class I and class II major histocompatibility complexes, or MHC) which present antigens on the surface of cells. Cytotoxic T cells are induced to proliferate by specialized antigen presenting cells such as macrophage and dendritic cells which present antigenic peptides on their cellular surfaces in conjunction with MHC molecules. T cells are induced by these antigen presenting cells to recognize corresponding antigens expressed on MHC antigens on the surface of target cells. T cells destroy these target cells.
The T cell recognizes the antigen in the form of a polypeptide fragment bound to the MHC class I molecules on target cells, rather than the intact polypeptide itself. The polypeptide is endogenously synthesized by the cell, and a portion of the polypeptide is degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit xcex22 microglobulin. The peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific T cells. Investigations of the crystal structure of the human MHC class I molecule HLA-A2.1 indicate that a peptide binding groove is created by the folding of the xcex11 and xcex12 domains of the class I heavy chain (Bjorkman et al., (1987) Nature 329:506. Falk et al., (1991) Nature 351:290 have developed an approach to characterize naturally processed peptides bound to class I molecules. Other investigators have successfully achieved direct amino acid sequencing of the more abundant antigenic peptides in various HPLC fractions by conventional automated sequencing of peptides eluted from class I molecules (Jardetzky, et al. (1991) Nature 353:326 and mass spectrometry Hunt, et al., Science 225:1261 (1992). A review of the characterization of naturally processed peptides in MHC Class I is found in Rotzschke and Falk (1991) Immunol. Today 12:447.
Target T cells recognizing antigenic peptides can be induced to differentiate and proliferate in response to antigen presenting cells bearing antigenic peptides in the context of MHC class I and class II complexes. There are differences in the antigenic peptides bound to MHC class I and class II molecules, but the two classes of bound peptides share common epitopes within the same protein which enable a T cell activated by an antigen presenting cell to recognize a corresponding MHC class I epitope. MHC class I molecules on target cells typically bind 9 amino acid antigenic peptides, while corresponding MHC class II-peptide complexes have greater heterogeneity in the size of the bound antigenic peptide.
The generation of target T cells with a desired specificity has been limited by the ability of investigators to discover appropriate peptides for loading onto MHC molecules, and by investigator""s ability to load peptide antigens onto antigen presenting cells used to induce proliferation of the T cells. In the past, investigators have generated antigen presenting cells by stripping the antigenic peptides normally found on antigen presenting cells by chemical or thermal techniques, followed by a reloading of the cells with a desired antigenic peptide. This approach has had limited success, due to inefficiencies in antigen presenting cell peptide loading, and due to the limited length of time that the loaded antigenic peptides remain loaded on the antigen presenting cells. In addition, only a single peptide fragment of a protein is loaded onto the surface of the antigen presenting cell using typical methods; thus, peptides important for activation of T cells against a target cell can be overlooked. The present invention overcomes these and other problems.
The invention provides new methods of making recombinant antigen presenting dendritic cells (DCs), which have been very difficult to transduce using existing methods. These new methods are applicable to the transduction of DCs with any recombinant nucleic acid. Also provided are new ways of expressing antigenic peptides on MHC molecules on the surface of the dendritic cells. It was surprisingly discovered that these expressed antigenic peptides are processed and displayed on the surface of the dendritic cells in the context of class I and class II MHC. These recombinant cells expressing antigenic peptides were found to be competent to activate T-cells against target cells expressing selected antigens in vivo. This provides powerful new treatments for cancers and cellular infections, as well as a variety of diagnostic and cell screening assays.
Naturally occurring dendritic cells are antigen presenting cells which activate T cell proliferation against target cells. Target cells express antigenic peptides in the context of MHC class I molecules on the surface of the target cell. Dendritic cells express related antigenic peptides on class I and class II MHC molecules. In a preferred use of the invention, dendritic cells are transformed with a nucleic acid encoding a heterologous protein which has a peptide subsequence corresponding to an antigenic peptide expressed on the surface of a target cell (on an MHC class I receptor). Preferably, a full-length protein is expressed, and several processed subsequences subsequently presented by the dendritic cell.
Surprisingly, heterologous proteins are expressed in the dendritic cell, processed into fragments, and expressed on the surface of the dendritic cell in the context of MHC class I and II molecules, making the dendritic cells capable of activating T cell proliferation against a target cell expressing the corresponding antigen. It is further demonstrated herein that T-cells activated by the dendritic cells of the invention by the methods of the invention are effective against established tumors and metastasis, in vivo. Thus, the present invention provides powerful new anti-cancer therapies based upon immunizing a patient with a recombinant dendritic cell, and/or T cell activated by a recombinant dendritic cell.
The new methods of transforming dendritic cells and expressing antigenic peptides on the surface of the cell to make the dendritic cell competent for T cell activation, provide significant advantages over prior art methods of loading peptides onto dendritic cells, including broader antigen expression and more efficient MHC class I and class II peptide loading, and the ability to expand the population of desired DCs, e.g., in culture. The invention has diagnostic, therapeutic and drug discovery assay uses.
DCs can be transduced with essentially any nucleic acid using the techniques provided. In one preferred embodiment, nucleic acids encoding cytokines (e.g., GM-CSF, an interleukin (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, etc.), or cell receptor ligands (e.g., transferrin, c-kit, viral receptor ligands, cytokine receptors, and the like) are transduced into stem cells to produce recombinant DC.
In one class of embodiments, the invention provides methods of transducing dendritic cells with selected nucleic acids. In the methods, a hematopoietic stem cell, e.g., a human CD34+ stem cell, is transduced with a selected nucleic acid, and the stem cell is then differentiated into a dendritic cell. Typically, the stem cell is differentiated in vitro using appropriate cytokines. For instance, mouse stem cells are differentiated into dendritic cells by incubating the stem cells in culture with murine GM-CSF. Typically, the concentration of GM-CSF in culture is at least about 0.2 ng/ml, and preferably at least about 1 ng/ml. Often the range will be between about 20 ng/ml and 200 ng/ml. In many preferred embodiments, the dose will be about 100 ng/ml. When human cells are transduced, human GM-CSF is used in similar ranges, and TNF-xcex1 is also added to facilitate differentiation. TNF-xcex1 is also typically added in about the same ranges. Optionally, SCF is added in similar dose ranges to make human DCs. Optionally, IL-4 is added in similar ranges, particularly for making murine DCs.
Ordinarily, the differentiation process is performed in vitro. Other cytokines such as IL-4 are optionally added to facilitate cell culture and cell differentiation. In addition, lipofectamine, or a similar transduction facilitating agent, is optionally added for improving gene transfer to cultures for producing recombinant DCs.
One preferred way of transducing a hematopoietic stem cell with a selected nucleic acid is to incubate the stem cell with a retroviral vector comprising the selected nucleic acid. Preferred vectors for stem cells include murine leukemia virus vectors. For human stem cells, murine leukemia virus vectors expressing Gibbon Ape leukemia virus envelopes are also preferred. For transducing murine stem cells, ecotropic envelopes are preferred.
Thus, the invention also provides recombinant dendritic cells with expression cassettes. The expression cassettes express proteins (or peptide fragments thereof) which are processed into antigenic peptides expressed on the surface of the dendritic MHC class I and II surface receptors. The expression cassettes typically comprise a strong promoter such as a t-RNA pol III promoter, or a pol II promoter with strong constitutive expression. One preferred pol II promoter is the retroviral murine leukemia virus LTR promoter. Example antigenic proteins expressed by the expression cassette include HER-2, MART-1, gp-100 and CEA, tyrosinase, MAGE, trp-1 and PSA.
In another preferred class of embodiments, the invention provides methods for activating T cells. In the methods, the T cell is contacted with a recombinant dendritic cell expressing a recombinant protein which is processed into antigenic peptides on the surface of the dendritic cell. The T cell is optionally contacted with the dendritic cell in vitro or in vivo. Thus, in one preferred embodiment, T cells are isolated from a mammal and incubated with recombinant dendritic cells in vitro. After incubation, the T cells can be used in assays, or re-introduced into the mammal to target and kill cells with antigenic peptides (bound to class I MHC molecules) corresponding to the peptides expressed on the surface of the dendritic cell. In another preferred embodiment, the recombinant dendritic cell is introduced into a mammal to activate the T cell in vivo. Preferred target cells are those expressing antigenic peptides in the context of MHC class I molecules, including cancer cells (e.g., prostate, colon, melanoma, and breast cancer cells), virally infected cells such as cells infected with an HIV, hepatitis or herpes virus, and parasitized cells including cells with intracellular bacterial infections and cells infected with parasites such as stages of P. falciparum (the primary causative agent for malaria).
The invention provides commercially valuable drug and cell assays. For instance, methods for detecting T cell mediated anti-cancer cell activity of a protein or peptide are provided. In the assays, a dendritic cell is transformed with a recombinant expression cassette encoding a heterologous protein or fragment thereof (e.g., an antigenic peptide) by the methods described herein. The T cell is contacted with the dendritic cell in vivo or in vitro, thereby activating the T cell against cells expressing a peptide antigen (on a MHC class I molecule) corresponding to an antigen expressed on the surface of the dendritic cell. To test whether the T cell has anti-cancer cell activity, a selected cancer cell (for instance a breast cancer, melanoma, prostate cancer, or colon cancer cell) is incubated with the T-cell (in vitro or in vivo) and inhibition of cancer cell replication, or T-cell mediated cancer cell lysis, or specific cytokine release (e.g., GM-CSF, IFN-xcex3 or TNF-xcex1) is observed. The assay is optionally performed in vitro, or optionally in vivo. By providing a way of discriminating proteins which can be targeted on cancer cells, the invention provides a commercially valuable assay. The same strategy can be applied to detect antigens or virally or parasitically infected cells by substitution of these cells for the cancer cells in the assay.
The activated T cells of the invention are generally cytotoxic against cells expressing antigenic peptides in the context of MHC which correspond to antigens expressed on antigen presenting cells. Thus, the invention provides a method for making T cells cytotoxic to selected target cells. In the methods, T cells are activated by contact with the recombinant dendritic cells of the invention, in vitro or in vivo.
The transformation of dendritic cells with target proteins changes the antigenic repertoire of the dendritic cell by causing processed peptide fragments to be expressed on the MHC molecules of the dendritic cell. Unlike untransformed dendritic cells, the recombinant transformed dendritic cells have processed peptide fragments derived from the target protein expressed on the surface of the dendritic cell.
In one embodiment, diagnostic assays are provided. These assays are used to determine whether a cell population (e.g., a blood or cell sample from a patient) express a selected antigen. In the assays, recombinant dendritic cells expressing the selected antigen are used to activate T-cells against the antigen. The cell population is then exposed to the activated T-cells, and lysis of the cells is monitored (e.g., by Trypan blue exclusion). If the observed lysis is higher than an appropriate control, the population of cells comprises the antigen. This can be used, e.g., to assess whether tumor cells express a particular antigen. In another class of diagnostic assays, the invention provides a way of monitoring precursor frequency and/or T-cell reactivity by exposure to recombinant DCs. This is an indicator of the effect of immunization with DCs.